Evaluation of peracid formation as the basis for resistance to infection in plants transformed with haloperoxidase.

Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxidation of alkyl acids to peracids. Among acids tested as substrates, acetic acid was most readily peroxidized. The reaction product peracetate possessed potent antifungal activity: 50% death (LD(50)) of Aspergillus flavus occurred at 25 microM peracetate. Viability of A. flavus was inhibited by up to 80% by leaf extracts of tobacco plants transformed with the HPO-P gene from P. pyrrocinia compared to viability of fungi exposed to extracts from controls. To elucidate if peracid formation by HPO-P was the basis for antifungal activity in transgenic leaf tissues, lethalities of hydrogen peroxide-acetate-HPO-P combinations against A. flavus were examined in vitro. LD(50) of A. flavus exposed to the combinations occurred at 30 mM acetate when concentrations of hydrogen peroxide and HPO-P were held constant. This value was identical to the LD(50) produced by 30 mM acetate in the absence of hydrogen peroxide-HPO-P and therefore did not account for enhanced antifungal activity in transgenic plants. For clarification, kinetics of the enzymic reaction were examined. According to the concentration of acetate needed for enzyme saturation (K(m) = 250 mM), acetate was lethal prior to its oxidation to peracetate. Results indicate that peracid generation by HPO-P was not the basis for enhanced antifungal activity in transgenic plants expressing the HPO-P gene.

All-D-cecropin B: synthesis, conformation, lipopolysaccharide binding, and antibacterial activity.

Cecropin B (LCB) is a natural peptide with antibacterial and antifungal properties. The enantiomer of LCB, containing all-D amino acids (DCB), was synthesized to examine its antibacterial and binding properties. The conformation of DCB was compared to its enantiomer by circular dichroism. Both the L- and D-peptides showed an identical induction of alpha-helical secondary structure. However, binding studies between Lipopolysaccharide (LPS) and DCB or LCB were studied with a dimethylmethylene blue spectrophotometric assay, showing the two enantiomeric peptides differed in their interaction with LPS. Antibacterial activity of DCB was determined against three Gram-negative bacteria, Pantoea agglomerans (ATCC 27996), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 17648), giving comparable results to LCB.

Antifungal and peroxidative activities of nonheme chloroperoxidase in relation to transgenic plant protection.

Nonheme chloroperoxidase (CPO-P) of Pseudomonas pyrrocinia catalyzes the oxidation of alkyl acids to peracids by hydrogen peroxide. Alkyl peracids possess potent antifungal activity as found with peracetate: 50% killing (LD(50)) of Aspergillus flavus occurred at 25 microM compared to 3.0 mM for the hydrogen peroxide substrate. To evaluate whether CPO-P could protect plants from fungal infection, tobacco was transformed with a gene for CPO-P from P. pyrrocinia and assayed for antifungal activity. Leaf extracts from transformed plants inhibited growth of A. flavus by up to 100%, and levels of inhibition were quantitatively correlated to the amounts of CPO-P activity expressed in leaves. To clarify if the peroxidative activity of CPO-P could be the basis for the increased resistance, the antifungal activity of the purified enzyme was investigated. The LD(50) of hydrogen peroxide combined with CPO-P occurred at 2.0 mM against A. flavus. Because this value was too small to account for the enhanced antifungal activity of transgenic plants, the kinetics of the enzyme reaction was examined and it was found that the concentration of hydrogen peroxide needed for enzyme saturation (K(m) = 5.9 mM) was already lethal. Thus, the peroxidative activity of CPO-P is not the basis for antifungal activity or enhanced resistance in transgenic plants expressing the gene.

D-cecropin B: proteolytic resistance, lethality for pathogenic fungi and binding properties.

L-Cecropin B (LCB) is a potent fungicidal peptide that is subject to proteolytic degradation by extracellular enzymes produced by Aspergillus flavus. We hypothesized that D-cecropin B (DCB), containing all D-amino acids, should resist proteolysis while retaining its fungicidal and target specificities. DCB was synthesized by solid phase methods using Fmoc chemistry. In vitro, at pH 6 x 0, DCB was lethal against the germinating conidia of A. flavus (LD90, 25 microM) and A. fumigatus (LD98, 25 microM) and for nongerminating and germinating conidia of Fusarium moniliforme (LD98, 1 x 25 microM) and F. oxysporum (LD95, 2 x 5 microM) at concentrations similar to those previously reported for LCB. It was lethal for Candida albicans with an LD98 at 12 x 5, microM. DCB was not active for the nongerminating conidia of A. fumigatus or A. flavus. Papain, trypsin, pepsin A and Staphylococcus aureus V8 protease degraded LCB but not DCB. Binding assays and circular dichroism showed DCB and LCB bound to cholesterol, ergosterol, beta-1,3-glucan, mannan and chitin. Data show that DCB retains the potent fungicidal properties of the L-form while being resistant to proteolytic enzymes that degrade the latter peptide. This study demonstrates that D-enantiomerization of cecropin B yields a novel fungicidal peptide, which resists proteolytic degradation and is lethal for pathogenic fungi.

Inhibition of fungal growth in planta and in vitro by transgenic tobacco expressing a bacterial nonheme chloroperoxidase gene.

Transgenic tobacco plants producing chloroperoxidase (CPO-P), encoded by a novel gene from Pseudomonas pyrrocinia, were obtained by Agrobacterium-mediated transformation. Successful transformation was shown by PCR, Southern, northern and western blot analyses, and assays of CPO-P enzyme activity. Extracts from plants transformed with the CPO-P gene significantly reduced Aspergillus flavus colonies by up to 100% compared with extracts from control plants transformed with pBI121. Compared with controls, the transformed plants showed increased disease resistance in planta against a fungal pathogen, Colletotrichum destructivum, the causal agent of tobacco anthracnose.

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores.

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.

Fungal lethality, binding, and cytotoxicity of syringomycin-E.

Syringomycin-E (SE) was significantly lethal to Aspergillus and Fusarium species at between 1.9 and 7.8 micrograms/ml. SE complexed with the following fungal wall constituents (in order of binding): beta-1,3-glucan > chitin > mannan > ergosterol = cholesterol. Cytotoxicity in HeLa cells was proportional to the SE concentration, while the amount required for cytotoxicity was 3 to 20 times that needed to kill 95% of the fungi tested.

Fungicidal properties, sterol binding, and proteolytic resistance of the synthetic peptide D4E1.

The fungicidal properties of the synthetic peptide D4E1 were studied with nongerminated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium moniliforme, and Fusarium oxysporum. The minimal lethal concentrations (MLC) needed to kill 100% of germinating conidia of A. fumigatus, A. flavus, and A. niger were 12.5, 12.5, and 25 microM, respectively. The MLC value for nongerminated and germinating conidia of both Fusarium spp. was 3.0 microM. Except for A. fumigatus, D4E1 was inactive against the nongerminated conidia of the Aspergillus spp. Physicochemical studies showed D4E1 complexed with ergosterol, a sterol present in conidial walls. Cholesterol, present in nongerminated conidia of F. moniliforme, had a greater affinity for D4E1 than did ergosterol. D4E1 was more resistant to fungal and plant protease degradation than the natural peptide, cecropin A. These in vitro results suggest D4E1 is a candidate for transgenic expression in plants to enhance host resistance to fungal infection.

Fungicidal and binding properties of the natural peptides cecropin B and dermaseptin.

In vitro fungicidal properties of cecropin B and dermaseptin were explored using non-germinating and germinating conidia from Aspergillus flavus, A. fumigatus, A. niger, Fusarium n2oniliforme and F oxysporum. Cecropin B produced LD50 values for germinating A. flavus, A. fumigatus and A. niger conidia of 30, 0.5 and 2.0 microM, respectively, while dermaseptin gave LD50 values of 4.0, 0.05 and 2.0 microM, respectively. Cecropin B produced an LD50 value of 0.2 microM for non-germinating F. moniliforme and F. oxysporum conidia, while dermaseptin did not reduce either as much as 50% at any level tested. LD50 levels for CB were 0.2 and 0.1 microM, respectively, for germinating F. moniliforme and F. oxysporum conidia. Dermaseptin was less effective, giving LD50 values for germinating F. moniliforme and F. oxysporum conidia of 0.3 and 0.8 microM, respectively. Neither peptide reduced conidial viabilities of non-germinating Aspergillus spp. Physicochemical studies indicated cecropin B and dermaseptin bound to ergosterol and cholesterol, conidial wall constituents, but not to chitin or beta-1,3-glucan.

Fungicidal and binding properties of three plant peptides.

The fungicidal properties of plant seed peptides from Heuchera sanginea (Hs-AFP1), Raphanus sativus (EA-AFP2), and Impatiens balsamina (Ib-AMP3) were determined for the non-germinated and germinated conidia of Aspergillus flavus and Fusarium moniliforme. These peptides were weakly lethal for germinated but not for non-germinated conidia of A. flavus. Both non-germinated and germinated conidia of F. moniliforme were susceptible to these peptides. Overall, F. moniliforme was more susceptible than A. flavus to the peptides. The peptides bound strongly to chitin, mannan, galactocerebrosides, and sphingomyelin. Binding results varied for ergosterol, cholesterol, and beta1,3-glucan.

Fungicidal activity of cecropin A.

Cecropin A (CA) fungicidal properties were explored. Nongerminated and germinated Aspergillus spp. and Fusarium spp. conidia were treated with CA. CA achieved complete lethality at < or = 25 microM (99 micrograms/ml) for germinating, but not nongerminating, conidia of Aspergillus spp. CA achieved total lethality for nongerminated and germinating conidia of Fusarium spp at 1.5 microM (6 micrograms/ml). MIC and minimal lethal concentration assays in buffered RPMI medium gave similar results.

Superoxide, hydrogen peroxide, and the respiratory burst of fungally infected plant cells.

The principal route of oxygen utilization in the respiratory burst of fungally infected plants was determined from stoichiometries of the uptake and electronic reduction of oxygen in cotton cells exposed to Aspergillus flavus walls. Using 2,2'-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) and epinephrine as redox reagents to manipulate oxygen transitions, we found that oxygen consumption doubled when superoxide disproportionation was abolished and was abolished when disproportionation doubled. Of four possible pathways for oxygen consumption, only monovalent reduction of molecular oxygen to superoxide was consistent with this inversely proportional relationship. According to the observed rate of oxygen consumption in this pathway and in the absence of competition to disproportionation of superoxide, infected cells are capable of generating intracellular concentrations of 1 M hydrogen peroxide in 13 min.

Binding between lipopolysaccharide and cecropin A.

Cecropin A (CA), a bioactive peptide, produced significant lethality to Pantoea agglomerans (PA) at low concentrations. Significant mortality occurred immediately after addition of CA. Separate preincubations of lipopolysaccharides (LPS) from the following bacteria: PA, Serratia marcescens, Escherichia coli (EC), and Salmonella typhimurium with CA were performed prior to the bioassay. CA was also preincubated with diphosphoryl lipid A (DPL-A) from EC and S. minnesota (SM), trilinolein, palmitic, lauric and myristic acids (fatty acids contained in the lipid A of PA-LPS) and bovine brain gangliosides. Spectral analysis to determine the interaction between glycosphingolipids (sphingomyelin, bovine brain gangliosides, and galactocerebrosides) and CA were performed. Results showed that all types of LPS and DPL-A as well as gangliosides studied blocked CA lethality to PA. The level of inhibition of CA antibacterial properties was dependent on LPS and DPL-A concentration. The individual fatty acids and trilinolein did not affect CA lethality to PA. Spectral studies showed complexation between CA and PA-LPS, both types of DPL-A, and the glycosphingolipids. Biological and chemical analyses confirm that CA binds to the diphosphoryl lipid A moiety of LPS.

Permanent gases inside healthy and microbially infected cotton fruit during development.

Permanent gases inside developing cotton fruit (Gossypium hirsutum L.) were, by weight, 46% nitrogen, 29% oxygen, 4% argon and 20% carbon dioxide, whereas plant canopy air assayed at 73% nitrogen, 25% oxygen, 2% argon and 0.3% carbon dioxide. Light exposure, fruit age, and mild infection (Erwinia) had no compositional effect but aggressive infection (Aspergillus) raised carbon dioxide content to 31% by weight and correspondingly lowered oxygen to 17%. Respiration with oxygen replenishment except during aggressive infection accounted for the fruit gas compositions.

Potential of animal myeloperoxidase to protect plants from pathogens.

The effect of animal myeloperoxidase (EC 1.11.1.7) on the viability of a plant pathogen was determined. Lethality of hydrogen peroxide to germinating spores of Aspergillus flavus increased 90-fold enzymically. Singlet oxygen was present but hypochlorite accounted for two-thirds of the increase. The results indicate myeloperoxidase could improve microbial resistance in plants, perhaps transgenically.

Relationship of byssinosis to the generation of oxygen radicals by bract tissues of cotton plants.

Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to bysinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanisms. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed. We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.

Substrate depletion during formation of aflatoxin and kojic acid on corn inoculated with Aspergillus flavus.

Depletion of sugar and starch carbon sources and concomitant formation of secondary fungal metabolites, aflatoxin and kojic acid, were examined in growing corn inoculated with Aspergillus flavus. Kernels from control and inoculated ears were removed and analyzed after 16, 24, 48, 96 and 168 hrs. Reducing sugars were not significantly different for inoculated and control non-inoculated samples, but after 168 hrs (seven days) starch content was 20% lower in inoculated than in control samples. Kojic acid was detected before aflatoxins formed. Kojic acid, the oxidized product of kojic acid, and aflatoxin were all present in samples two days from inoculation. The formation of this oxidation product may influence toxin levels.

Aflatoxin Estimation in Corn by Measurement of Bright Greenish-Yellow Fluorescence in Aqueous Extracts.

A procedure was developed for efficient extraction of bright greenish-yellow (BGY) fluorescent material from ground corn ( Zea mays L.) samples and subsequent quantitative determination of fluorescence emission in a fluorometer. The technique was designed to be inexpensive, rapid and utilizable at remote locations for estimation of aflatoxin levels. A simple, in vitro method for production of the BGY-fluorescing material from kojic acid was developed to provide a reference standard. Water was identified as an effective solvent for extraction of the BGY-fluorescence from corn; fluorescence in aqueous extracts was stable at pH 6-7 for up to 22 h at room temperature. Samples with aflatoxin concentrations of 0 to 12,000 ppb yielded BGY-fluorescence emission units of 0 to 175. A correlation coefficient of r = 0.77 was determined for BGY-fluorescence vs total aflatoxin levels (B1, B2, G1, G2) and r = 0.88 for the fluorescence vs aflatoxin B1, B2. The results provided a basis for effective determination of BGY-fluorescence as an estimate of aflatoxin levels in corn.